The present application expressly incorporates by reference U.S. Ser. No. 08/383,180, now U.S. Pat. No. 5,721,209, U.S. Ser. No. 08/796,791, now U.S. Pat. No. 5,786,326, U.S. Ser. No. 08/882,122; and U.S. Ser. No. 08/960,714, all to Horwitz, et al., all of which are subject to an exclusive license or assignment to the same assignee as this application.
The present invention relates to a heretofore undisclosed process for the synthetic generation of high affinity, iron binding compounds known as Exochelins, and more particularly, to a synthetic process for making Exochelins and to modifications to these newly synthesized compounds to vary their physiological properties, including applications of these newly synthesized and utile compounds for diagnosing and treating disease in mammals.
The above referenced U.S. Patents and applications have shown that exochelins have unique physiological benefits. For example, in acute myocardial infarction, cardiac tissue is damaged by two sequential events, hypoxia in the ischemic phase and oxidative damage in the reperfusion phase. Myocardium damaged in the ischemic phase can be salvaged by reintroduction of blood into the ischemic area. However, reperfusion can result in injury to the reperfused tissue as a result of an inflammatory response caused by the migration of leucocytes into the tissue and the production of reactive oxygen species. One of the most reactive species is the hydroxyl radical (xe2x80x94OH) which is generated in the presence of iron, and which often results in cell death or related oxidative tissue damage.
Prevention of the formation of (xe2x80x94OH) prevents lethal cell damage by several mechanisms. It is well known that the formation of (xe2x80x94OH) is dependent on the presence of free iron, and that iron chelators will prevent reperfusion injury. For example, the iron chelator deferoxamine, when administered prior to reperfusion, prevents injury and reduces myocardial infarct size during coronary artery occlusion and reperfusion. However, reperfusion injury occurs rapidly after the reestablishment of blood flow to the ischemic myocardium.
The formation of the (xe2x80x94OH) radical is dependent on the presence of free iron and iron chelators can scavenge the free iron and thus render the iron unavailable to catalyze the hydroxyl radical formation. However, prior known chelating means either do not prevent (xe2x80x94OH) production by the Fenton Reaction (i.e., EDTA), or enter the cells too slowly (i.e., desferrioxamine). As a result, sufficient quantities of the chelating agent are not available to act rapidly enough to chelate enough iron to prevent the formation of (xe2x80x94OH) and cell damage and destruction which results.
Desferrioxamine has been demonstrated to be effective if administered prior to occurrence of the myocardial infarct but to be ineffective if administered at or after the onset of reperfusion. Similar injury to heart tissue can occur as a result of heart bypass procedures, such as during open heart surgery, or to other body organs when they are deprived of oxygenated blood as a result of surgery or injury. Thus, iron scavenging chelators are clearly needed to prevent oxidative tissue damage.
Prior to the discoveries of Horwitz, et al., compounds referred to as Exochelins had been briefly described, and their general function in the growth of mycobacteria was likewise discussed by Macham, Ratledge and Barclay at the University of Hull in England (MACHAM, L. P., RATLEDGE, C. and NOCTON J. C., xe2x80x9cExtracellular Iron Acquisition by Mycobacteria: Role of the Exochelins and Evidence Against the Participation of Mycobactinxe2x80x9d, Infection and Immunity, December 1975, pp.1242-1251, Vol. 12, No.6; BARCLAY, R. and RATLEDGE, C., xe2x80x9cMycobactins and Exochelins of Mycobacterium tuberculosis, M. bovis, M. africanum and Other Related Speciesxe2x80x9d, Journal of General Microbiology, 1988, pp.134, 771-776; MACHAM, L. P. and RATLEDGE, C., Journal of General Microbiology, 1975, pp. 89, 379-382).
Macham identified the existence of a substance found in the extracellular fluid, which he referred to as xe2x80x98exochelinxe2x80x99. Macham further described the materials he referred to as exochelins as water and chloroform soluble compounds having the ability to chelate free iron. Macham, et al., did not isolate or purify the compounds, merely characterizing them as penta- or hexapeptide, with molecular weights in the range of 750 to 800, inter alia.
According to Macham""s work, his exochelins have similarities to mycobactinxe2x80x94which is located in the cell wall and functions to transmit iron to the interior of the cell. However, unlike mycobactin, a lipophilic, water insoluble molecule which is unable to diffuse into, and assimilate free iron from the extracellular environment, Exochelin functions at physiological pH to sequester iron from other iron bearing compounds in the serum. Also, depending on the bacterial source of the compounds, Macham, et al. disclosed that his molecules may also include salicylic acid or beta-alanine.
Barclay et al. (Ibid.)likewise described the production of compounds he called exochelins from twenty-two different strains of M. tuberculosis and related species. However, neither these, nor any other known prior investigators, determined the specific structure of those compounds, or identified any application for the same outside of their function as a transport medium for iron to mycobactin located in the cell wall.
In sum, Macham et al. recognized that after sequestering iron from, for example, ferritin or transferrin (and the like iron bearing compounds found in the serum) his compounds present the iron in a form that can be transferred to mycobactin, while Barclay et al. described production of his compounds from known mycobacterial strains without precisely elucidating their structure.
The total synthesis of a related compound, Mycobactin S2, was reported by Maurer and Miller in 1983 (MAUER, P. J. and MILLER, M. J. xe2x80x9cTotal Synthesis of a Mycobactin: Mycobactin S2xe2x80x9d, 1983, J. Am. Chem. Soc., pp. 240-245, Vol. 105). Mauer et al. successfully prepared 29 milligrams of a Mycobactin utilizing a complex, multi-step synthetic pathway. Mycobactin S2, however, is significantly different from the target molecule according to the synthesis of the present invention. Likewise, Exochelin synthesis remains unreported to date.
The following references provide teachings relevant to the synthesis according to the present invention:
MAUER, P. J. and MILLER, M. J., 1982, J. Am. Chem. Soc., 104, 3096;
FARKAS, L. et al., 1967, Tetrahedron, 23, 741;
SCHNIEPP, L. E. and GELLER, H. H., 1946, J.Am. Chem. Soc., 68, 1646;
GAUDRY, R., 1948, Can. J. Res. Sect. B, 26, 387;
DREYFUSS, P., 1974, J. Med. Chem., 17(2),252;
BERLINGUET, L. and GAUDRY, R., 1952, J. Biol. Chem., 198, 765;
BODANSZKY, M., et al., 1978, J. Med. Chem., 21(10),1030;
MAURER, P. J. and MILLER, M. J. 1981, J. Org. Chem. Soc., 46(13), 2835;
BIRNBAUM, S. M., LEVINTOW, L. KINGSLEY, R. B. and GREENSTEIN, J. P., 1952, J. Biol. Chem., 194, 455;
COREY, E. J. and VANKATESWARLU, A., 1972, J. Am. Chem. Soc., 94, 6190;
SIEBER, P., 1977, Helv. Chim. Acta, 60, 2711 (b);
GERLACH, H., 1977, Helv. Chim. Acta, 60, 3039;
MITSUNOBU, O., 1981, Synthesis, 1981, 1.
Horwitz, et al., have discovered the currently accepted structural nature of exochelins, and patented uses of the same as novel iron chelators to inhibit the iron mediated oxidant injury which occurs during reperfusion, and have applications pending to other hydroxyl radical related insults to living tissues, including cancer, arteriosclerosis, organ preservation and vessel occlusion following angioplasty.
Likewise, the synthesis of related compounds strongly suggests the medical need for, and the production of synthetic versions of such important and needed compounds. See, for example, HU, J. and MILLER, M. J., xe2x80x9cTotal Synthesis of a Mycobactin S, a Siderophore and Growth Promoter of Mycobacterium Smegmatis, and Determination of its Growth Inhibitory Activity against Mycobacterium tuberculosisxe2x80x9d, 1997, pp.3462-3468, J. Chem. Soc. 119. However, complications in synthesizing the desired compounds require modifications to known procedures, and various sterochemical constraints have prevented generating Exochelins through synthetic routes.
With chelation of iron now being recognized as a means for preventing the oxidative damage of living tissue, the potential applications for Exochelins and related compounds abound. As an iron scavenger in a physiological system capable of withdrawing iron from iron-bearing proteins, Exochelins effectively prevent cell destruction following interruption of blood flow. Similarly, chelation of other metals can regulate levels of the same in various other therapeutic settings, including the delivery of various desirable metals to the body, or the targeting of diseased organs with beneficial drugs bound to exochelins, or the like synthetic transport means.
Prior work of Horwitz, et al., resulted in purified Exochelins and demonstrated their utility as scavengers of free iron and their effectiveness in preventing the formation of tissue damaging hydroxyl radicals. In particular, Horwitz, et al., purified Exochelins from M. tuberculosis and demonstrated that they effectively removed iron from transferrin, lactoferrin and ferritin at physiological pH, without transmitting any of the infectious properties of the tuberculosis bacteria. Likewise, Horwitz, et al., were responsible for showing for the first time that these Exochelins block hydroxyl radical formation by the Fenton reaction and, based upon the response of cardiac myocytes, are effective for preventing reperfusion injury after myocardial infarction or vascular insults to other tissue when administered after an attack occurs, in addition to several hours following such an episode.
Further, Horwitz, et al., in eludicating the chemical structure of Exochelins noted that prior references cited above had failed to define the actual structure and, instead, characterizing the exochelins as peptides. These unsuccessful attempts to identify the actual structure of the exochelin family likewise have hindered anyone from undertaking or accomplishing their synthesis. By uncovering the broad range of molecular weights which Exochelins have, Horwitz, et al., have discovered that several series of compounds with identifiable differences in molecular weights are properly included with the grouping. Exochelins cannot be considered to be peptides; instead they contain three amino acids and other structural moieties (salicylic acid, dicarboxylic acids or monoester analogs, and hydroxy carboxylic acids) formed by amide (xe2x80x94NHxe2x80x94COxe2x80x94), hydroxymate (xe2x80x94NH(OH)xe2x80x94COxe2x80x94) and ester condensations (xe2x80x94COxe2x80x94O).
Likewise, in copending U.S. Ser. No. 08/882,122 deprivation of iron has been shown to attack cancer cells by modes which are particularly well addressed by Exochelins. Owing to their very high affinity for iron and their lipid solubility, Exochelins produced from, Mycobacterium tuberculosis possess enhanced ability to enter cells. A synthetic iron chelator with lipid solubility clearly would help to address cancer diagnosis, treatment, and screening as well as other physiological problems addressed by use of biologically derived exochelins.
Clearly, there exists a longstanding need for an improved synthetic agent or compound effective for rapidly chelating metals as they become available, to counteract myocardial infarction, and treat cancer and other related medical conditions driven by the presence of free metals, or protect tissue which may be damaged by the hydroxyl radical and related mechanisms imparting cell death and destruction.
Accordingly, it is an object of the invention to provide a process for the synthetic preparation of Exochelins which overcomes the drawbacks of the prior art. An additional object of the invention is to synthesize a compound which behaves, in all respects, like the natural material isolated by Horwitz, et al.
An additional object of the present invention is to provide a process to prepare synthetic Desferri Exochelin 772SM(R) or (S) which rapidly chelates ferric iron in solution.
It is a further object of the invention to provide for the chemical synthesis of Desferri-Exochelin 772SM(R) or (S) effective for rapidly binding iron, among other metals.
It is an additional object of the present invention to provide a synthetically prepared Exochelin compound that elutes on reverse-phase HPLC (phenyl column) at the same concentration of acetonitrile as the native molecule.
It is yet still another object of the invention to provide a simplified process design for the generation of synthetic Exochelins on an industrial scale for use in the prevention of oxidative damage to living tissue.
Briefly stated, there is provided a synthetic process for generating Exochelin 772SM(R), a new molecule having a seven carbon chain terminating in a methyl ester coupled to an acyclic hydroxamate, with four stereogenic centers, including three S-isoforms and an R-configuration at a B subunit.
According to a feature of the present invention there is provided, a process for the synthesis of EXOCHELIN 772SM(R) comprising the steps of reacting a mixture of pimelic acid, dimethyl pimelate, hydrochloric acid, methanol and di-n-butyl ether to produce methyl hydrogen pimelate and then mixing the methyl hydrogen pimelate with thionyl chloride and dimethyl formamide to generate methylpimeloyl chloride which was stored for later reaction. This was then added to a suspension of O-benzyl hydroxylamine hydrochloride and triethyl amine in CH2Cl2 to produce O-benzylmethylpimelyl hydroxamate. To a solution of (L)-6-hydroxynorleucine and triethylamine in a tetrahydrofuran (THF)-water mixture a solution of di-tert-butyl dicarbonate in THF was added. An aqueous layer was then acidified to a pH 3 with citric acid and extracted with EtOAc. The organic layer was dried and purified to produce (L)-N-Boc-6-hydroxynorleucine (L)-N-Boc-6-hydroxynorleucine was reacted with allyl bromide to produce (L)-N-Boc-6-hydroxynorleucine allyl ester. To (L)-N-Boc-6-hydroxynorleucine allyl ester and carbon tetrabromide in anhydrous CH2Cl2 was added triphenylphosphine to provide a viscous oil, which was then added to EtOAc/hexane and (L)-N-Boc-6-bromonorleucine allyl ester was recovered. A mixture of (L)-N-Boc-6-bromonorleucine allyl ester, O-benzylmethylpimelyl hydroxamate, potassium iodide (Kl) and potassium carbonate in anhydrous acetone was prepared and (L)-N6-methylpimelyl-N6-(benzyloxy)-N2-Boc-lysine allyl ester was recovered. Trifluoro acetic acid was added to the (L)-N6-methylpimelyl-N6-(benzyloxy)-N2-Boc-lysine allyl ester to form a solid intermediate which was added to (L)-N-(2-(benzyloxy)benzoyl)serine and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline to produce a light brown viscous oil identified as (L)-N6-Methylpimelyl-N6-(benzyloxy)-N2-((L)-N-(2-(benzyloxy)benzoyl)serine)-lysine allyl ester. Thionyl chloride was gradually added to a cooled (xe2x88x9230xc2x0 C. bath) solution of the lysine allyl ester in anhydrous tetrahydrofuran (THF) and the golden colored oil was then purified to provide a light brown viscous oil identified as (L)-N6-Methylpimelyl-N6-(benzyloxy)-N2-((S)-2-(2-benzyloxy)phenyl)-2-oxazoline-4-carbonyl)-lysine allyl ester. To the solution of oxazoline-lysine allyl ester in anhydrous CH2Cl2 was added morpholine and tetrakis(triphenylphosphine)palladium to provide an acid. To a solution of acid and (L)-Nxcex1-((S)-3-hydroxybutyryl)-xcex1-amino-N-(benzyloxy)caprolactam in anhydrous THF was gradually added diethyl azodicarboxylate. The material isolated was mixed with MeOH, palladium and hydrogen. The mixture was filtered and the solvent was subsequently removed under reduced pressure and co-evaporated with CH2Cl2 to provide an off-white flaky solid determined by NMR analysis indicated to be Exochelin 772SM(R) shown as formula 1a.
The above, and other objects, features and advantages of the present invention will become apparent from the following description read in conjunction with the accompanying figures, tables and formulae, outlining and describing a set of embodiments of synthetic processes according to the present invention, wherein like reference designating numerals are employed throughout to represent common chemical species moieties and functional groups.